History and Accomplishments

Research and development on many projects has continued in the division of molecular pathology.

Diagnostic Testing Research Project

WHAT HAS BEEN ACCOMPLISHED

Research and development on many projects has continued in the division of molecular pathology. New clinical tests have been validated and implemented in the area of infectious disease, and development of additional tests has begun-such as the division’s rapid response to develop, optimize and implement a molecular assay for the H1N1 influenza outbreak of 2009. This effort was an important tool in the hospital’s triage of the sickest patients during the pandemic’s most crucial time. With the continued vital support from the Grant-A-Starr Foundation, progress on multiple projects was achieved including the following:

VIRAL INFECTIONS

Herpes simplex virus (HSV) is a cause of viral meningitis and life-threatening encephalitis in children. Rapid diagnosis of central nervous system infections associated with herpes simplex viruses will save lives because these infections are treatable with antiviral agents. In addition to testing from cerebrospinal fluid (CSF), testing for HSV-1 and HSV-2 is available from blood as well, which prevents the need for painful and difficult spinal taps in our tiniest patients.

Enterovirus testing from CSF is an important tool in the diagnosis of meningitis. Development of a rapid test or this pathogen is underway. Further optimization will include parallel testing of enterovirus and HSV from one CSF collection (meaning one spinal tap only for older patients where CSF is still the most accurate method for diagnosis) as well as enterovirus testing from stool.

Respiratory viruses are the most common cause of acute illness in the U.S. and cause life- threatening infections in young children. Current laboratory testing capabilities include: respiratory syncytial virus, influenza, parainfluenza, and human metapneumovirus.

The 2009 H1N1 outbreak presented a critical situation for Texas Children’s. A real-time PCR assay that tested for influenza A and H1N1 influenza was rapidly developed and put into practice. This massive effort provided results to our physicians in mere days compared to the weeks until the CDC confirmation results were available. Bacterial infections

Testing for Bartonella henselae, the bacterium responsible for cat-scratch fever in children, is available from tissue samples.

Expansion of our DNA pyrosequencing capabilities has allowed for speciation of Burkholderia cepacia complex organisms. A gene specific to this genus is now being sequenced in order to positively identify organisms within the complex, thus providingour physicians with information that may alter their treatment approach.

Under collaboration with commercial partners, several multi-pathogen panels are underevaluation and development including panels capable of detecting important pathogens in sepsis, gastrointestinal infections, and meningitis/encephalitis.

THE PROJECT TEAM EMPLOYS THE USE OF MANY TECHNOLOGIES, INCLUDING THE FOLLOWING:

RNA and DNA Extraction

DNA Pyrosequencing

Real-Time PCR

Liquid Bead Arrays

DNA Chips or Glass-based Microarrays

THE MILESTONES AND OUTCOMES

YEAR 1 - DISCOVERY AND DEVELOPMENT

The project team will focus on shifting the scope of the project to DNA sequencing methodologies, including optimization of DNA/RNA extraction and sequencing methods for a variety of specimen types important in the diagnosis of sepsis, gastroenteritis and meningitis. At least three candidate pathogens of new human microbes/viruses will be identified, and approval of human studies protocols from the institutional review board will be obtained.

YEAR 2 - CONTINUED DISCOVERY AND DEVELOPMENT

Discovery and development efforts will continue with the sequencing of a minimum of 100 clinical samples. Sequencing data analysis tools and algorithms will be developed, and a large scale hospital-based microbe/virus/pathogen sequence database will be established. Development will include the addition of specimen types vital to pneumonia and early-detection sepsis as well as the introduction of droplet PCR to boost throughput potential. By the end of Year 2, roughly 625 megabases of sequencing data (1 megabase equals 1 million bases) will have been produced, testing from a minimum of five specimen types will have been optimized, and the initial work will have been submitted for presentation at national scientific meetings.

YEAR 3 - BEGIN DELIVERY OF DIAGNOSTIC SERVICES

Delivery of new diagnostic tests will begin in Year 3 with the introduction of the first next generation sequencing test in the inpatient pediatric setting. New tests will continue to be developed, and tissue samples will be added to the growing specimen optimization list. By the end of Year 3, the project will have produced 2.5 gigabases of sequencing data (1 gigabase equals 1 billion bases) from over 200 clinical samples, at least 1,000 microbe sequences will have been added to the sequence database, and the first journal article will be submitted for potential publication.

YEAR 4 - MILESTONES

Implementation of new diagnostic tests continues through Year 4, with new tests continuing to be added to the development pipeline based on the discoveries made in the prior years. New specimen types for optimization will also be added at this time. By the end of Year 4, a total of 5 gigabases of sequencing data (1 gigabase equals 1 billion bases) will have been produced from over 400 clinical samples, and at least 1,500 microbe sequences will have been added to the sequence database. It is expected that a minimum of four new diagnostic sequencing applications will have been validated for use at Texas Children’s with at least six additional tests in development and 20 new candidate pathogens/microbes/viruses in the discovery/development phase.

YEAR 5 - MILESTONES

The final year of the timeline includes continued progress in all three areas. By the end of Year 5, over 10 gigabases of sequencing data (1 gigabase equals 1 billion bases) will have been produced, with at least 2,000 sequenced microbes from 600 clinical samples added to sequence database. At least 30 new candidate pathogens/microbes/viruses will have been identified, and at least 10 new sequencing tests will be in development and moving towards implementation.